European Journal of Human Genetics (2014), 1–8
& 2014 Macmillan Publishers Limited All rights reserved 1018-4813/14
www.nature.com/ejhg
ARTICLE
Homozygous loss of DIAPH1 is a novel cause
of microcephaly in humans
A Gulhan Ercan-Sencicek1,2, Samira Jambi3,16, Daniel Franjic4,16, Sayoko Nishimura1,2, Mingfeng Li4,
Paul El-Fishawy1, Thomas M Morgan5, Stephan J Sanders6, Kaya Bilguvar2,7, Mohnish Suri8,
Michele H Johnson9, Abha R Gupta1,10, Zafer Yuksel11, Shrikant Mane7,12, Elena Grigorenko1,13,
Marina Picciotto1,4,14, Arthur S Alberts15, Murat Gunel2, Nenad Šestan4 and Matthew W State*,6
The combination of family-based linkage analysis with high-throughput sequencing is a powerful approach to identifying rare
genetic variants that contribute to genetically heterogeneous syndromes. Using parametric multipoint linkage analysis and whole
exome sequencing, we have identified a gene responsible for microcephaly (MCP), severe visual impairment, intellectual
disability, and short stature through the mapping of a homozygous nonsense alteration in a multiply-affected consanguineous
family. This gene, DIAPH1, encodes the mammalian Diaphanous-related formin (mDia1), a member of the diaphanous-related
formin family of Rho effector proteins. Upon the activation of GTP-bound Rho, mDia1 generates linear actin filaments in the
maintenance of polarity during adhesion, migration, and division in immune cells and neuroepithelial cells, and in driving
tangential migration of cortical interneurons in the rodent. Here, we show that patients with a homozygous nonsense DIAPH1
alteration (p.Gln778*) have MCP as well as reduced height and weight. diap1 (mDia1 knockout (KO))-deficient mice have
grossly normal body and brain size. However, our histological analysis of diap1 KO mouse coronal brain sections at early and
postnatal stages shows unilateral ventricular enlargement, indicating that this mutant mouse shows both important similarities
as well as differences with human pathology. We also found that mDia1 protein is expressed in human neuronal precursor cells
during mitotic cell division and has a major impact in the regulation of spindle formation and cell division.
European Journal of Human Genetics advance online publication, 30 April 2014; doi:10.1038/ejhg.2014.82
INTRODUCTION
Microcephaly (MCP) is a neurodevelopmental disorder characterized
by a small cranium with a significantly reduced occipito-frontal head
circumference. The reduced brain volume is particularly evident
within the cerebral cortex and is often associated with some degree
of intellectual disability (ID). MCP is categorized as either primary
(MCP vera) or syndromic, indicating the presence of other physical
or neurological deficits or gross structural brain abnormalities.
Mendelian forms of MCP have been identified, representing both
dominant and recessive forms of inheritance.1–3 In the present
study, we report a consanguineous Saudi Arabian pedigree
with a novel syndromic form of MCP, involving small head size,
ID, seizures, short stature, and blindness. Genome-wide linkage
analysis identified a maximum LOD score of 3.7 under a recessive
model of inheritance mapping to chromosome 5q31.3.
Whole-exome sequencing (WES) and targeted sequencing
analyses identified a rare, nonsense, homozygous sequence
variant c.2332C4T (p.Gln778*, RefSeq NM_005219.4) in the
gene DIAPH1 (mouse symbol is Diap1) (MIM 602121) encoding
the mammalian diaphanous-related formin, mDia1. The encoded
protein has previously been implicated in neuronal migration
of cortical interneurons,4 autosomal hearing loss,5 and
myelodysplasia.6 Depending upon the cell type and position in
the cell cycle, mDia1 has been shown to localize to the cell cortex,
trafficking endosomes, cleavage furrow, mid-bodies, and
centrosomes, the cytoplasmic microtubule-organizing center
crucial for cell division.7 These properties are shared with several
other primary microcephaly genes, including WDR62 (Homo
sapiens WD repeat domain 62),2,8,9 CENPJ (centromere protein J),10
CEP152 (centrosomal protein 152 kDa),11 ASPM (abnormal spindlelike MCP-associated (Drosophila)),12 and CEP135 (centrosomal
protein 135 kDa).13 Interestingly, analysis of Diap1-deficient mice
revealed cerebral ventricular enlargement but not the reduction in
brain volume and blindness observed in the human phenotype. Thus,
DIAPH1 has a crucial role in brain development and exhibits species
differences in its function.
MATERIALS AND METHODS
The study protocol was approved by the Yale Human Investigation Committee
(HIC). Approval from the institutional review board for genetic studies and
1Child Study Center, Yale University School of Medicine, New Haven, CT, USA; 2Department of Neurosurgery, Program on Neurogenetics, Yale School of Medicine, New Haven,
CT, USA; 3Al Hada Armed Hospitals, Taif, Saudi Arabia; 4Department of Neurobiology and Kavli Institute for Neuroscience, Yale School of Medicine, New Haven, CT, USA;
5Department of Pediatrics, Vanderbilt University, Nashville, TN, USA; 6Department of Psychiatry, University of California, School of Medicine, San Francisco, CA, USA; 7Yale
Center for Mendelian Genomics, New Haven, CT, USA; 8Nottingham Clinical Genetics Service, Nottingham University Hospitals NHS, Trust City Hospital Campus, Nottingham,
UK; 9Departments of Diagnostic Radiology, Surgery and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA; 10Department of Pediatrics, Yale University
School of Medicine, New Haven, CT, USA; 11Department of Medical Genetics, Osmangazi University, School of Medicine, Eskisehir, Turkey; 12Department of Genetics, Yale
University School of Medicine, New Haven, CT, USA; 13Moscow State University for Psychology and Education, Moscow, Russia; 14Departments of Psychiatry, Pharmocology
Yale University School of Medicine, New Haven, CT, USA; 15Laboratory of Cell Structure and Signal Integration, Van Andel Research Institute, Grand Rapids, MI, USA
*Correspondence: Dr MW State, Department of Psychiatry, University of California, School of Medicine, San Francisco, CA 94143-0984, USA. Tel: +1 415 476 7730;
Fax: +1 415 476 7320; E-mail: [email protected]
16These authors contributed equally to this work.
Received 16 October 2013; revised 16 March 2014; accepted 4 April 2014
Loss of function mutation in DIAPH1
AG Ercan-Sencicek et al
2
informed written consent from the family were obtained by the referring
physician in the Alhada Military Hospital, Taif, Saudi Arabia. Nucleotide
numbers are in reference to cDNA (NM_005219.4 where A of the ATG
translational start site is designated as þ 1) and amino acid number is in
reference to protein (O60610) coordinates.
incubated with mouse anti-mDia1 (1:50, Santa Cruz-sc-373807, Dallas, TX,
USA) and rabbit anti-pericentrin (1:500, Covance-PRB-432C, Princeton, NJ,
USA). Alexa Fluor 488 Donkey Anti-Mouse IgG and Alexa Fluor 594 Donkey
Anti-Rabbit IgG were used as secondary antibodies (Invitrogen, Grand Island,
NY, USA). DNA was visualized with DAPI. Cells were imaged with an Aperio
microscope (Aperio Technologies, Vista, CA, USA).
Neuroimaging
Magnetic resonance images (MRIs) containing axial, sagittal, and coronal
TI- and T2-weighted images were obtained from patient IV:7 in this pedigree.
The corpus callosum, posterior fossa structures (the cerebellum, midbrain,
pons, and medulla), deep gray matter, degree of myelination, and presence of
any associated malformations were assessed on each MRI.
Genotyping, targeted sequencing, and WES
Genomic DNA derived from whole blood was genotyped using the 1Mv1 Duo
Bead array chips (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. SNPs showing Mendelian inconsistency in transmission
were removed14 and data were pruned to 42 703 SNPs that are in minimal
linkage equilibrium with each other using linkage disequilibrium (LD)-based
SNP pruning with the following criteria: pairwise correlation coefficient
r2o0.23 within a 50-SNP window; proportion of missing genotypes o5%,
relative minor allele frequency 45%, and Hardy–Weinberg equilibrium 0.0001
among the controls and cases. Family relatedness was ascertained through
estimation of identity by descent. The LOD score was calculated with pruned
SNPs using Allegro 1.2 (DeCode genetics Inc., Reykjavik, Iceland).
DNA from both patient IV:4 and the unaffected father were sequenced using a
custom-designed NimbleGen Sequence Capture 385K array (460 bp probes to
sequence the target linkage region; Roche Nimblegen, Inc., Madison, WI, USA)
and later data from patient IV:4 using WES according to the manufacturer’s
protocol. WES was conducted as previously described.2 The sequence reads were
mapped to the human genome (NCBI/hg19). Variants were confirmed by
Sanger sequencing. DIAPH1 sequence alteration and other variations
(Supplementary Tables 4 and 6) found in patient IV:4 were submitted to the
LOVD public database (http://databases.lovd.nl/shared/individuals/00004216).
DIAPH1 screening in independent cases and control groups
All 28 exons comprising DIAPH1 were evaluated in the following individuals:
(1) 62 Turkish and Middle Eastern subjects found to have regions of
homozygosity corresponding to chromosome 5q31, presenting one or more
of the following conditions: MCP, lissencephaly, polymicrogyria, cerebellar
hypoplasia, schizencephaly, corpus callosum hypogenesis, demyelination,
psychosomatic motor delay, ID, epilepsy, and autism; (2) 136 Turkish
probands, including 120 from consanguineous and 16 from non-consanguineous families presenting with MCP; and (3) 100 neurologically normal
control individuals from Saudi Arabia. PCR primer sequences and conditions
are available upon request.
Quantitative PCR and western blot analysis
DIAPH1 mRNA expression in Epstein–Barr virus (EBV)-transformed lymphoblastoid cells (LCLs) available from four affected individuals and both parents
was assessed by real-time-PCR (RT-PCR) using DIAPH1 and the reference
gene TBP (TATA box-binding protein) primers with the PCR efficiency of
490% (slope ¼ 3.2 and 3.6) and R2 99%. Relative changes in gene
expression were analyzed with the DCT method.15 Cell lysates from LCLs of
four affected individuals, parents, and an independent control were used for
western blot analysis. We used primary rabbit anti-DIAPH1 antibodies
from N-terminus (1:10 000, Abcam-ab11172, Cambridge, MA, USA) and
C-terminus (1:5000, Bethyl Labs-A300–078, Montgomery, TX, USA).
Anti-GAPDH (1:200) was used as the loading control.
Cellular localization of mDia1
ReNcell CX cells from the immortalized fetal human cortical neural progenitor
cell line (Millipore-SCC007, Billerica, MA, USA) were fixed in 4% PFA and
blocked by blocking solution (5% normal donkey serum, 1% BSA, 0.1%
glycine, 0.1% lysine, and 0.3% Triton X-100 in PBS). Fixed cells were
European Journal of Human Genetics
Human and mouse brain specimens
The study protocol was approved by the HIC. All human tissues were collected
under guidelines approved by the Yale HIC and were part of the tissue
collection at the Department of Neurobiology. For each tissue donation,
appropriate written, informed consent and approval were obtained. All
experiments with mice were carried out in accordance with protocols approved
by the Institutional Animal Care and Use Committee at Yale University, School
of Medicine. diap1-knockout mice are a gift from A S Alberts (Van Andel
Institute, MI, USA). The genotype of the diap1-deficient allele was analyzed
by PCR using the following primers: Wt F-50 -TAGATAGGGATAAAGTTG
GTGGGCTGTG-30 , Wt R-50 -GAGATGTCCTGCAATGACTGAGCAGTGG-30 ,
and Mut R-50 -GCATCACCTTCACCCTCTCCACTGACAG-3’. The morning of
a detectable vaginal plug and the first neonatal day were considered to be
embryonic day 0.5 (E0.5) and postnatal day 0 (P0), respectively.
In situ hybridization
For embryonic stages, pregnant females were anesthetized, and pups at E12.5,
E14.5, and E17.5 stages were extracted from the uterus. Embryonic brains were
dissected and fixed by immersion in 4% PFA. At all postnatal stages (P3, P7,
P14), mice were anesthetized with injectable anesthetics and intracardially
perfused with 4% PFA. Brains were postfixed overnight in 30% sucrose/4%
PFA, and sectioned in the coronal plane on a Leica sledge cryomicrotome at
36 mm. Sections were processed for nonradioactive in situ hybridization.
The RNA probes complementary to mouse diap1 (bases 3062–3861 of the
mouse diap1 cDNA, NM_007858.2) and human DIAPH1 (bases 2241–3960
of the human DIAPH1 cDNA, NM_005219.4) were labeled with digoxigenin11-UTP. Sections were analyzed with a Zeiss Stemi dissecting microscope or a
Zeiss AxioImager with an AxioCam MRc5 digital camera (Zeiss, Oberkochen,
Germany).
Immunostaining
For immunostaining, fixed E14.5 and P0 brain tissues were embedded in 4%
agarose and sectioned at 50 mm with a Vibratome VT1000S (Leica, Wetzlar,
Germany). Tissue sections were incubated in primary antibodies
(Supplementary Table 5) and with appropriate donkey Alexa Fluor-conjugated
secondary antibodies (1:1000, Invitrogen). Nissl staining was performed using
the standard method.
Gene co-expression network
To identify any possible correlation of the DIAPH1 gene with MCP genes
(MCPH1 (MIM 607117), WDR62 (MIM 613583), CDK5RAP2 (MIM 608201),
ASPM (MIM 605481), CENPJ (MIM 609279), STIL (MIM 181590), and
CEP135 (MIM 611423)), we performed co-expression analyses using the
previously generated human brain transcriptome data set.16 For each MCP
gene, we used Pearson’s correlation analysis with other genes, and then chose
its top 100 highly correlated genes. Cytoscape software17 was used to visualize
the co-expression network, in which genes are shown by circles and the
correlated genes are connected by lines. The ‘un-weighted force-directed
layout’ parameter was used to optimize the network visualization. The
human brain data set is generated by exon arrays and is available from the
Human Brain Transcriptome database and the NCBI Gene Expression
Omnibus under accession number GSE25219. It covers 16 brain regions
over 15 periods, ranging in stage from embryonic development to late
adulthood.16 To test the significance of overlapping eight genes between
DIAPH1 and CDK5RAP2, permutation analysis was performed. Briefly, we
randomly picked two groups with 100 genes in the whole human gene list. We
set up the threshold that the number of genes shared by these two groups
should be larger than eight. We repeated this operation 10 000 000 times and
Loss of function mutation in DIAPH1
AG Ercan-Sencicek et al
3
counted the number of operations that passed the preset threshold.
The P value was estimated by the ratio of successful operations.
RESULTS
A new MCP syndrome with temporal pole atrophy, callossal
hypotrophy, seizure, and blindness
We ascertained a consanguineous family from Saudi Arabia (Pedigree
SAR1008) including five (three female and two male) children that
came to clinical attention initially at B3 months of age because of
seizures and were found to have severe MCP (head circumferences
more than 3 SD below the mean for age) (Figure 1a and Supplementary Data).
The mother had a history of a single second-trimester miscarriage
(IV:1) prior to delivering her first-born child (IV:2). It is not known
whether the fetus had MCP. The second offspring (IV:3) was full term,
but died at 3 months of age, despite hospitalization in a NICU,
II:1
because of congenital heart disease. In addition, patient IV:5 recently
died at the age of 18 as a result of chest infection.
Clinical examinations of the four oldest affected individuals showed
moderate to severe ID, delays in both motor and language development, and severe bilateral visual impairment. Their hearing was
grossly normal bilaterally, as was audiological examination. The
youngest affected individual, patient IV:8, was assessed at 3 months
and 3 weeks of age. He was found to be microcephalic (head
circumference less than 3rd percentile); his weight was 5 kg (3rd
percentile), and he suffered from seizures and bilateral visual
impairment. Additional details of the medical history are presented
in the Supplementary data.
T1- and T2 weighted, non-contrast, 1.5T MR imaging revealed that
the brain of affected individual IV-7 exhibited a substantial reduction
in the size of the cerebral cortex without obvious evidence of
abnormal neuronal migration or grossly abnormal architecture
I:1
I:2
II:2
II:3
*
*
III:2
III:1
*
*
IV:1
IV:2
IV:4
T2
*
*
IV:5
IV:6
*
*
IV:7
T1
IV:8
T1
Control
T2
IV:3
II:4
IV:7
r
s
on
on
oc
Figure 1 The pedigree structure and radiographic features of the SAR1008 family. (a) The patients described in this study are numbered. Parents are first
cousins and have three affected female (IV:4, IV:5, IV:6), two affected male (IV:7, IV:8) and one unaffected female (IV:2) children. Squares and circles
indicate males and females, respectively. Affected individuals are shown as filled symbols. One pregnancy that resulted in miscarriage is shown as a
triangle. A diagonal line through a symbol denotes that the individual is deceased. The individuals from whom DNAs were obtained are indicated by *.
(b) Representative MRIs of individual IV:7 at 4 months of age and control subject at 3 years of age. Axial T2 and T1 at the level of the temporal lobe,
T2 and T1 at the level of the chiasm, and T1 sagittal images are shown (left to right). The MRI reveals temporal pole atrophy with normal cortical thickness.
The optic nerves (on) and chiasm (oc) appear normal in size (indicated by arrows). Midline sagittal T1 shows hypoplasia of the rostrum (r) and splenium (s)
of the corpus callosum. The scale bars are in cm.
European Journal of Human Genetics
Loss of function mutation in DIAPH1
AG Ercan-Sencicek et al
4
(MCP vera). He also had volume loss of the anterior end of the
temporal lobe (the temporal pole) with normal cortical thickness
suggesting atrophy rather than hypoplasia. There was also loss of
volume of the rostrum and splenium of the corpus callosum on
midline sagittal T1-weighted images. The optic nerves and chiasm,
cerebral aqueduct, and fourth ventricle were normal on midline
sagittal T1 (Figure 1b). The subject’s small weight and height
indicated that body size was also affected.
A homozygous nonsense sequence variant in DAPH1 causes MCP
We performed whole-genome genotyping of all members of the
pedigree SAR1008 and confirmed the reported parental relationship
of first cousin (found pi-hat ¼ 0.15, consistent with a first-cousin
union) (Supplementary Table 1). We then performed parametric
linkage analysis using both affected and unaffected individuals and
specifying a recessive mode of inheritance, penetrance of 99%, a
phenocopy rate of 0.01, and a disease allele frequency of 0.0001 using
a pruned set of 42 703 SNPs selected on the basis of LD information
derived from the Saudi Arabian and Middle Eastern populations.14
We identified a single linkage peak on chromosome 5q31.3 reaching
the maximal theoretical LOD score of 3.7 under the specified
parameters. This locus is flanked by SNPs rs2907308 and rs164078
and constitutes an approximately 724 kb region containing 38
annotated genes (Figure 2a and Supplementary Table 2). No other
region of the genome was found to reach genome-wide significance
(Supplementary Figure 1).
Next, in order to identify the disease-causing sequence variant,
using the gDNA from proband IV:4, we performed WES as well as
targeted sequencing of the LOD-2 confidence interval (138.8–160 cM),
achieving a very high level of coverage (Supplementary Table 3).
For targeted sequencing, we also used the gDNA from the father,
focusing on the LOD-2 confidence interval (138.8–160 cM), using a
custom-designed NimbleGen Sequence Capture 385K array based on
the Illumina Genome Analyzer IIx platform.
Within the targeted linkage interval, we identified only a single
homozygous, nonsense sequence variant affecting the DIAPH1 gene.
No other novel homozygous coding insertion or deletion sequence
variants were observed within this interval. We identified a total of 15
homozygous missense variants that were reported previously at
dbSNP with their minor allele frequencies between 0.086 and
47.2% (Supplementary Table 4). The nonsense sequence variant in
DIAPH1, c.2332C4T (r.2473c4u), was predicted to terminate
translation at amino acid 778 of 1272 (p.Gln778*, Q778*), leading
to a truncated 86 kDa protein lacking the carboxyl-terminus bearing
Figure 2 Identification of a nonsense homozygous sequence variant in the DIAPH1 gene in a family with MCP. (a) Panel shows the results of the parametric
linkage analysis for chromosome 5q, expressed as LOD scores. The maximum theoretical LOD score for this family is 3.7 using the genotyping data from
IMv1 Duo Bead array chips. The analysis is modeled under the recessive mode of inheritance, penetrance of 99%, a phenocopy rate of 0.01, and a disease
allele frequency of 0.0001. (b) Domain organization of mDia1. Abbreviations: GBD, GTPase binding region; DID, diaphanous inhibitory domain; DD,
dimerization domain; CC, coiled coil; FH1, formin homology 1 domain; FH2, formin homology 2 domain; DAD, diaphanous autoinhibitory domain. Nonsense
sequence variant p.Gln778* results in a 86.2 kDa truncated protein. (c) mRNA levels of DIAPH1 gene in EBV transformed LCLs from homozygous patients
(IV:4, IV:5, IV:6, IV:7), and heterozygous parents (III:1 and III:2) were analyzed using RT-PCR. Three individuals of the same ethnicity and without a
sequence variant are used as control (WT). (d) Protein blot for mDia1. Lane 1 represents the control subject, lanes 2 and 3 represent the unaffected
parents who carry the heterozygous p.Gln778* alteration (III:1 and III:2). Lanes 4, 5, 6, and 7 represent affected individuals with homozygous p.Gln778*
alteration (IV:4, IV:5, IV:6, IV:7). The band indicates a molecular weight of B140 kDa. GAPDH is used as control (lower band).
European Journal of Human Genetics
Loss of function mutation in DIAPH1
AG Ercan-Sencicek et al
5
the highly conserved formin homology (FH2) domain that is
responsible for mDia1’s ability to generate linear actin filaments
and affect microtubule dynamics (Figure 2b and Supplementary
Figure 2A and B). The presence of the heterozygous sequence variant
in both parents was confirmed via Sanger sequencing. Further analysis
of WES data did not identify any other rare, segregating LoF (loss-offunction) homozygous sequence variants (Supplementary Table 6)
either within or outside the linkage interval.
We anticipated that the mutant allele would lead to nonsensemediated decay and evaluated the expression via qRT-PCR in the
index family. We found a four-fold decrease in DIAPH1 expression in
probands relative to their parental controls (Figure 2c). Subsequent
western blot analysis showed that all affected subjects lacked the
predicted 140 kDa mDia1 protein. The heterozygous parents showed
protein expression levels approximately one-half of that observed in
an unrelated control (Figure 2d).
Further Sanger sequencing of the complete coding and UTR
regions of the DIAPH1 gene in 200 control chromosomes from
neurologically normal individuals from Saudi Arabia, a cohort of 136
MCP cases, and 62 consanguineous cases that have homozygosity for
the interval surrounding DIAPH1 and at least one of the following
phenotypes, ID, seizure, MCP, lissencephaly, polymicrogyria, cerebellar
hypoplasia, and/or autism, failed to identify any additional rare,
homozygous LoF alterations. These findings strongly suggest that the
homozygous loss-of-function sequence variant identified in DIAPH1
and the accompanying absence of protein expression is the underlying
genetic cause of the disorder in the index family.
mDia1 is localized pericentrosomally in mitotic neural progenitors
To gain insight into the potential functions of mDia1 in brain
development, we analyzed Diap1 mRNA expression in the developing
mouse and human forebrain and in hNPCs. In situ hybridization of
mouse brain at embryonic days (E) 12.5, 14.5, and 17.5 revealed that
Diap1 mRNA was expressed in the ventricular and subventricular
zone (VZ and SVZ, respectively) progenitor cells of the dorsal and
ventral forebrain and the brainstem (Figures 3a and b). Diap1 was
also observed in neurons of the cortex and hippocampus of later
embryonic age (Supplementary Figure 3A). During postnatal development, Diap1 expression was detected in the cerebral cortex, basal
ganglia, hippocampus, thalamus, and external granular layer of the
Figure 3 Expression pattern and subcellular localization of DIAPH1 in E14.5-day-old mouse brain (this period of development is equivalent to human brain
12 PCW), fetal human brain, and mitotic human cortical neural progenitor cells. A set of two panels from a developing mouse brain (E14.5) indicates (a)
PAX6 (under the white line) at VZ, and (b) in situ hybridization shows Diap1 expression at VZ and SVZ. (c) In situ hybridization of early fetal human brain
(12 PCW) shows DIAPH1 expression in the VZ and SVZ. The scale bar indicates 100 mm and 600 mm, respectively. See Supplementary Figure 3 for
individual panels. (d) Confocal microscopy analysis of fixed human cerebral cortical neural progenitor cells in mitosis (M phase). Cells were co-stained with
anti-mDia1 (green), pericentrin (red), and DAPI for DNA (blue). The scale bar indicates 10 mM.
European Journal of Human Genetics
Loss of function mutation in DIAPH1
AG Ercan-Sencicek et al
6
cerebellum (Supplementary Figure 3B). Similar to mouse Diap1
expression, the human homolog was strongly expressed by VZ and
SVZ progenitor cells of the human neocortical wall at 12 weeks post
conception (PCW) (Figure 3c).
Given that the majority of MCP genes encode proteins that localize
to the centrosome or mitotic spindle, we hypothesized that mDia1
would show similar subcellular localization and tested this in hNPCs
using immunofluorescence. Double immunofluorescence staining for
mDia1 and pericentrin (PCNT), a centrosomal protein, revealed colocalization with centrosomes and mitotic spindles in mitosis
(M phase) (Figure 3d).
To gain insight into function we conducted mRNA co-expression
analyses to identify genes with similar spatial and temporal expression
pattern to DIAPH1 and other previously identified MCP genes in the
developing human brain. We used exon-array data derived from
human brain covering 16 brain regions over 15 periods, ranging from
embryonic development to late adulthood.16 The top 100 mostcorrelated transcripts of the seed genes, including the seven previously
identified MCP genes and DIAPH1, were chosen using Pearson’s
correlation analysis and visualized with Cytoscape. Using this
approach among the other MCP genes, we found that DIAPH1 and
CDK5RAP2 are correlated with each other by sharing eight
overlapping genes. To test the significance of this overlapping, we
performed a permutation test that showed that the overlapping
between DIAPH1 and CDK5RAP2 is significant with P-value o 1e 7,
suggesting spatio-temporal expression and potential functional
overlap between the two genes in particular (Supplementary
Figure 4). Together, these findings indicate that, similar to previously
identified MCPH genes, mDia1 is localized pericentrosomally in
mitotic neural progenitors and shares a number of functionally
related co-expressed genes during human brain development.
Mice lacking mDia1 have enlarged lateral ventricles but not MCP
In order to further evaluate the role of mDia1 in embryonic brain
development, we examined Diap1-targeted mice (Diap1 / ) lacking
mDia1 protein.18 We confirmed the absence of mDia1 protein in the
developing forebrain using western blot (Supplementary Figure 5).
Consistent with the previous study,19 we found that Diap1 / mice
show normal organization of the cerebral cortex using layer-specific
markers, CUX1, BCL11B (also known as CTIP2), and FOXP2 at P0
(Figures 4a and b and Supplementary Figure 6). However, our
analysis of tissue sections of Diap1 / mice with Nissl staining
revealed unilateral dilatation of the ventricles in 60% of mutant mice
(9 out of 15) with no blockage of the cerebral aqueduct (Figures 4c
and d, Supplementary Figures 7). Previous analyses of mice lacking
both DIAP1 and DIAP2 (mDia-DKO) had also various degrees of the
dilation of the lateral and the third ventricle in all adult mDia-DKO
mice and this was due to periventricular dysplasia in the third
ventricle resulting in obstruction of CSF circulation.19
Given that mDia1 nucleates and processively elongates linear
actin filaments20 and that the actin cytoskeleton has a critical
role in the regulation of adherence junctions in the
neuroepithelium, we also stained tissue sections of mice using
fluorescently labeled phalloidin and immunofluorescence with
antibodies against other adherent junction components such as
b-catenin (CTNNB1), N-cadherin (CDH2), and E-cadherin
(CDH1). We found that actin filaments smoothly lined the
apical surface of neuroepithelial progenitor cells in wild-type
Figure 4 Cortical organization is preserved in the Diap1 / mouse. Image depicts deeper cortical layer marker TBR1, layer VI (green), and phalloidin
staining in the coronal sections of the lateral ventricle wall in (a) Wt (Diap1 þ / þ ) and (b) mutant (Diap1 / ) mice at E14.5 day. Boxed areas are shown at
the higher magnification. Scale bars 200 mm at lower magnification and 50 mm at higher magnification. (c–d) Nissl staining of coronal brain sections of
Diap1-KO and Wt at P0. Note the lateral ventricle dilatation on the Diap1-KO mouse. Scale bar is 1 mm.
European Journal of Human Genetics
Loss of function mutation in DIAPH1
AG Ercan-Sencicek et al
7
and Diap1 / mice (Figure 4b and Supplementary Figure 8, upper
panel). Our result showed that, in contrast to the requirement for
both mDia1 and mDia3, as revealed by analysis of Diaph1/2-DKO,
loss of mDia1 alone does not affect the assembly of the apical actin
belt at the apical surface of neuroepithelial cells as well as the adhesion
molecules in the adherens junctions. mDia1 and other formins also
modulate the microtubule (MT) cytoskeleton.21,22 We investigated
a-tubulin and g-tubulin expression in Diap1 / animals at E14.5.
The absence of DIAPH1 did not grossly alter the organization of
tubulin in coronal sections of the lateral vertical wall (Supplementary
Figure 8, middle and lower panels).
Prior study of Diap1/2-null, but not Diap1-null, mice revealed
impaired tangential migration of cortical and olfactory interneurons
compared with either wild-type or Diap2-mutant mice at E16.5.4 Our
analysis of Diap1 / mice did not reveal any obvious alterations in
the distribution of GABA-containing interneurons in the cortex
(Supplementary Figure 9).
Given that affected human individuals exhibit hypoplasia of the
corpus callosum, we analyzed Diap1 / animals for changes in
callosal thickness. Comparison between Diap1 / and wild-type
littermates at P0 revealed no significant differences in cortical
white matter or callosal thickness (Supplementary Figure 10).
Together, these findings indicate that loss of function of Diap1 in
mice, unlike in humans, does not lead to MCP or callosal
hypoplasia.
DISCUSSION
We have identified a new human MCP gene, DIAPH1, in a
consanguineous family with syndromic congenital MCP, blindness,
seizures, developmental delay, ID, and short stature. mRNA and
protein expression studies confirm that, similar to other MCP genes,
DIAPH1 is expressed in neural progenitors where it is associated with
the centrosomes and mitotic spindle. Specifically, expression was
found within VZ and SVZ zones of the developing cerebral wall. The
centrosome is an organelle that has multiple critical roles in the
organization of the mitotic spindle and the astral microtubules during
mitosis and in neuronal migration to the cortical plate. Similar to
most MCP genes that encode proteins that localize to the centrosome
or mitotic spindle, the DIAPH1 gene product mDia1 showed perinuclear localization, which was concentrated on mitotic spindles and
co-localized with centrosomes in the M phase of hNPCs at mitotic
division. Hence, the expression pattern of DIAPH1 in the brain was
consistent with a role in neurogenesis and in regulation of the size of
the cerebral cortex. Moreover, previously it has been reported that
both ARP2/3- and mDia1-dependent actin polymerization is required
for centrosome separation in early Drosophila embryos23 and that
DIAPH1 localizes to the spindle microtubules of non-neuronal cells
during mitotic cell division.24
The SAR1008 pedigree described here presented with MCP and
growth retardations, sharing features with other syndromal microcephalies, including Seckel syndrome (MIM 210600)25 and MOPD II
(MIM 210720).26 Consistent with our current findings, another
centrosomal protein, PCNT, leads to Seckel and MOPD II
syndrome and has been shown necessary for efficient recruitment
of Cdk5rap2 to the centrosome in neural progenitors.27 As noted, our
co-expression analysis using known MCP genes identified a link
between DIAPH1 and CDK5RAP2 that is associated with centrosomal
function and mitotic spindle orientation. CDK5RAP2 localizes at the
centrosome during mitosis and interacts directly with microtubuleend binding protein (EB1) to regulate microtubule dynamics and
stability.28 Interestingly, EB1 and adenomatous polyposis coli (APC)
interact with the FH1 and FH2 domains of DIAPH1 to stabilize
microtubules and promote cell migration.29 This evidence supports a
model in which different MCP genes converge on the same
mechanism.
Although animal models have been quite useful for illuminating
cellular and molecular mechanisms of MCP, so far few have faithfully
and consistently recapitulated the human phenotype. For example,
the mouse model for Aspm, which was represented by two mouse
lines (Aspm1–25 and Aspm1–7), identified only mild reductions in
brain size in Aspm1–7, and no significant difference in brain size in
Aspm1–25.30 Similarly, a single study identified no significant difference
between Wt and KO-Mcph1 mice,31 whereas a second group showed
that Mcph1 disruption in mice resulted in MCP.32 In contrast, the
mouse model of Cdk5rap2 shows dramatic reductions in brain size
along with other brain phenotypes, including enlarged ventricles.33
In this case, Diap1 knockouts do not recapitulate a small brain
phenotype or callosal hypoplasia, but similar to Cdk5rap2 show
enlarged ventricles. These species differences may result from the
molecular and cellular differences in how the cerebral cortex develops
in the two species. Some of the most prominent features of cortical
neurogenesis in humans, when compared with mice, are the
prolonged neurogenesis,34 the elongation of the apical fiber of
radial glial cells (RGCs),35 and the enlargement of the outer SVZ
zone and outer RGCs.36,37 However, the most striking structural
finding in Diap1 KO mice was unilateral ventricular enlargement
(9 out of 15). Consistent with these findings, a variable degree of
ventricular enlargement has been reported as an MCP-related brain
malformation in both mice and humans.33,38
Recent published data from mouse KO models or non-neuronal
tumor cell lines in vitro suggest that mDia1 is a Rho-effector and
has a critical role in the maintenance of the adherens junction and
polarity of neuroepithelial cells in multiple brain regions and in
shaping F-actin dynamics that drives tangential migration of
neuronal precursors.4,19,39 Even though radial migration was
promoted and tangential migration was impaired in neuronal
precursors from Diaph1/2-DKO mice, this migration deficit is
more apparent for those migrating a longer distance of tangential
migration by regulating movement of F-actin condensation at the
leading edge and formation of an F-actin cup at the rear edge
of SVZ neural progenitor cells. Hence, the maximum distance
between centrosome and nucleus before nuclear translocation was
significantly reduced.4
Together, our genetic and molecular biological data, coupled with
previous findings, provide strong evidence that mDia1 has a crucial
role in brain development, leads to MCP in humans and exhibits
species differences in its function in the developing nervous system.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This study was supported by NIH grants; Clinical Investigation and the
Clinical and Translational Science (UL1 TR000142 and KL2 TR000140) from
the National Center for Research Resources (NCRR), a component of the NIH,
and NIH roadmap for Medical Research (PI; Robert Sherwin), NS054273,
MH081896 (PI; Nenad Sestan) and the Overlook International Foundation (to
MWS). ASA is grateful to the Jay and Betty Van Andel endowment and to Ms
Susan Kitchen-Goosen for Diap1 mutant mouse management. We thank the
families for their participation and AO Caglayan and Y Cheng (from M. Gunel
Lab) for technical assistance.
European Journal of Human Genetics
Loss of function mutation in DIAPH1
AG Ercan-Sencicek et al
8
1 Abuelo D: Microcephaly syndromes. Semin Pediatr Neurol 2007; 14: 118–127.
2 Bilguvar K, Ozturk AK, Louvi A et al: Whole-exome sequencing identifies recessive
WDR62 mutations in severe brain malformations. Nature 2010; 467: 207–210.
3 Thornton GK, Woods CG: Primary microcephaly: do all roads lead to Rome? Trends
Genet 2009; 25: 501–510.
4 Shinohara R, Thumkeo D, Kamijo H et al: A role for mDia, a Rho-regulated actin
nucleator, in tangential migration of interneuron precursors. Nat Neurosci 2012; 15:
S371–S372.
5 Lynch ED, Lee MK, Morrow JE, Welcsh PL, Leon PE, King MC: Nonsyndromic deafness
DFNA1 associated with mutation of a human homolog of the Drosophila gene
diaphanous. Science 1997; 278: 1315–1318.
6 Eisenmann KM, Dykema KJ, Matheson SF et al: 5q- myelodysplastic syndromes:
chromosome 5q genes direct a tumor-suppression network sensing actin dynamics.
Oncogene 2009; 28: 3429–3441.
7 Tominaga T, Sahai E, Chardin P, McCormick F, Courtneidge SA, Alberts AS:
Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling.
Mol Cell 2000; 5: 13–25.
8 Yu TW, Mochida GH, Tischfield DJ et al: Mutations in WDR62, encoding a centrosomeassociated protein, cause microcephaly with simplified gyri and abnormal cortical
architecture. Nat Genet 2010; 42: 1015–1020.
9 Nicholas AK, Khurshid M, Desir J et al: WDR62 is associated with the spindle pole and
is mutated in human microcephaly. Nat Genet 2010; 42: 1010–1014.
10 Bond J, Roberts E, Springell K et al: A centrosomal mechanism involving CDK5RAP2
and CENPJ controls brain size. Nat Genet 2005; 37: 353–355.
11 Guernsey DL, Jiang H, Hussin J et al: Mutations in centrosomal protein CEP152 in
primary microcephaly families linked to MCPH4. Am J Hum Genet 2010; 87: 40–51.
12 Bond J, Scott S, Hampshire DJ et al: Protein-truncating mutations in ASPM cause
variable reduction in brain size. Am J Hum Genet 2003; 73: 1170–1177.
13 Hussain MS, Baig SM, Neumann S et al: A truncating mutation of CEP135 causes
primary microcephaly and disturbed centrosomal function. Am J Hum Genet 2012;
90: 871–878.
14 Purcell S, Neale B, Todd-Brown K et al: PLINK: a tool set for whole-genome
association and population-based linkage analyses. Am J Hum Genet 2007; 81:
559–575.
15 Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C(T)
method. Nat Protoc 2008; 3: 1101–1108.
16 Kang HJ, Kawasawa YI, Cheng F et al: Spatio-temporal transcriptome of the human
brain. Nature 2011; 478: 483–489.
17 Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T: Cytoscape 2.8: new features for
data integration and network visualization. Bioinformatics 2011; 27: 431–432.
18 Peng J, Wallar BJ, Flanders A, Swiatek PJ, Alberts AS: Disruption of the Diaphanousrelated formin Drf1 gene encoding mDia1 reveals a role for Drf3 as an effector for
Cdc42. Curr Biol 2003; 13: 534–545.
19 Thumkeo D, Shinohara R, Watanabe K et al: Deficiency of mDia, an actin nucleator,
disrupts integrity of neuroepithelium and causes periventricular dysplasia. PLoS One
2011; 6: e25465.
20 Li F, Higgs HN: The mouse Formin mDia1 is a potent actin nucleation factor regulated
by autoinhibition. Curr Biol 2003; 13: 1335–1340.
21 Bartolini F, Gundersen GG: Formins and microtubules. Biochim Biophys Acta 2010;
1803: 164–173.
22 Palazzo A, Ackerman B, Gundersen GG: Cell biology: tubulin acetylation and cell
motility. Nature 2003; 421: 230.
23 Cao J, Crest J, Fasulo B, Sullivan W: Cortical actin dynamics facilitate early-stage
centrosome separation. Curr Biol 2010; 20: 770–776.
24 Kato T, Watanabe N, Morishima Y, Fujita A, Ishizaki T, Narumiya S: Localization of a
mammalian homolog of diaphanous, mDia1, to the mitotic spindle in HeLa cells. J Cell
Sci 2001; 114: 775–784.
25 Griffith E, Walker S, Martin CA et al: Mutations in pericentrin cause Seckel syndrome
with defective ATR-dependent DNA damage signaling. Nat Genet 2008; 40: 232–
236.
26 Rauch A, Thiel CT, Schindler D et al: Mutations in the pericentrin (PCNT) gene cause
primordial dwarfism. Science 2008; 319: 816–819.
27 Buchman JJ, Tseng HC, Zhou Y, Frank CL, Xie Z, Tsai LH: Cdk5rap2 interacts with
pericentrin to maintain the neural progenitor pool in the developing neocortex. Neuron
2010; 66: 386–402.
28 Fong KW, Hau SY, Kho YS, Jia Y, He L, Qi RZ: Interaction of CDK5RAP2 with EB1 to
track growing microtubule tips and to regulate microtubule dynamics. Mol Biol Cell
2009; 20: 3660–3670.
29 Wen Y, Eng CH, Schmoranzer J et al: EB1 and APC bind to mDia to stabilize
microtubules downstream of Rho and promote cell migration. Nat Cell Biol 2004; 6:
820–830.
30 Pulvers JN, Bryk J, Fish JL et al: Mutations in mouse Aspm (abnormal spindle-like
microcephaly associated) cause not only microcephaly but also major defects in the
germline. Proc Natl Acad Sci USA 2010; 107: 16595–16600.
31 Trimborn M, Ghani M, Walther DJ et al: Establishment of a mouse model with
misregulated chromosome condensation due to defective Mcph1 function. PLoS One
2010; 5: e9242.
32 Gruber R, Zhou Z, Sukchev M, Joerss T, Frappart PO, Wang ZQ: MCPH1 regulates the
neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry
through the Chk1-Cdc25 pathway. Nat Cell Biol 2011; 13: 1325–1334.
33 Lizarraga SB, Margossian SP, Harris MH et al: Cdk5rap2 regulates centrosome
function and chromosome segregation in neuronal progenitors. Development 2010;
137: 1907–1917.
34 Charvet CJ, Finlay BL: Embracing covariation in brain evolution: large brains, extended
development, and flexible primate social systems. Prog Brain Res 2012; 195: 71–87.
35 Rakic P: Evolution of the neocortex: a perspective from developmental biology. Nat Rev
Neurosci 2009; 10: 724–735.
36 Kennedy H, Dehay C: Self-organization and interareal networks in the primate cortex.
Prog Brain Res 2012; 195: 341–360.
37 Lui JH, Hansen DV, Kriegstein AR: Development and evolution of the human
neocortex. Cell 2011; 146: 18–36.
38 Passemard S, Titomanlio L, Elmaleh M et al: Expanding the clinical and neuroradiologic phenotype of primary microcephaly due to ASPM mutations. Neurology 2009;
73: 962–969.
39 Zaoui K, Honore S, Isnardon D, Braguer D, Badache A: Memo-RhoA-mDia1 signaling
controls microtubules, the actin network, and adhesion site formation in migrating
cells. J Cell Biol 2008; 183: 401–408.
Supplementary Information accompanies this paper on European Journal of Human Genetics website (http://www.nature.com/ejhg)
European Journal of Human Genetics
Supplementary Information
Homozygous loss of DIAPH1 is a novel cause of microcephaly in humans
A.Gulhan Ercan-Sencicek, Samira Jambi, Daniel Franjic, Sayoko Nishimura, Mingfeng Li, Paul
El-Fishawy, Thomas M. Morgan, Stephan J. Sanders, Kaya Bilguvar, Mohnish Suri, Michele H.
Johnson, Abha R. Gupta, Zafer Yuksel, Shrikant Mane, Elena Grigorenko, Marina Picciotto,
Arthur S. Alberts, Murat Gunel, Nenad Šestan, and Matthew W. State
Page
2
Clinical information about index family
Supplemental Figures and Tables
Figure S1.
Genome-wide parametric linkage analysis for SAR1008 pedigree
18
Figure S2.
The amino acid sequence alignment of DIAPH1 protein across species
using ClustalW2 program and nonsense homozygous mutation in DIAPH1.
19
Figure S3.
DIAPH1 Expression Pattern in Human and in embryonic and adult mouse
brains
20
Figure S4.
Gene co-expression analysis
21
Figure S5.
mDia1 expression in the whole brain lysates from wild-type (+/+), and
homozygous (–/–) mice of Diap1 genotype
22
Figure S6.
mDia1-KO mice show preserved layer organization
23
Figure S7.
Coronal sections of brain from Wt and mDia1-KO mice were stained with
Nissl
24
Figure S8.
Immunostaining for adherent junction components
25
Figure S9.
Immunostaining for GABA
26
Figure S10. Corpus callosum measurement
27
Table S1.
IBD estimates for parents and siblings
28
Table S2.
Genotyping data of the homozygous block on chromosome
29
Table S3.
Coverage distributions and error rates across the targeted homozygosity
interval
34
Table S4.
Variants identified within the homozygous linkage interval
35
Table S5.
Primary and secondary antibodies used for immunostaining
36
Supplemental References
37
1
Clinical information about index family
Family Birth Order and Miscarriage:
IV:1 (Miscarriage at 2nd trimester)
IV:2 (normal per family)
IV:3 (died at age of 3 months)
IV:4 (proband)
IV:5 (affected; died at age of 18 years old)
IV:6 (affected)
IV:7 (affected)
IV:8 (affected)
IV: 3 was born full term with congenital heart disease (according to hospital’s report) and he was
died at age of 3 months in neonatal intensive care unit (NICU). No other clinical information is
available about him.
Patient IV:4
Age at Interview: 15 years and 11 months
HPI: The parents have difficulty recalling exact dates and events. However, they believe that
they first became concerned when the patient started having seizures at three months of age.
Over the course of the first two years of her life, they also noticed abnormalities with her
development, including delays in language. In addition, they notice that she had significant
difficulties with vision.
Currently, they note poor adaptive skills that they attribute partly to her blindness and partly to
her decreased intellectual ability. They note that she cannot toilet or dress herself and that she
2
cannot feed herself. They say, however, that she can walk, sit, and explore objects with her
hands. They say that she does not have the intellectual capacity to attend school.
In terms of social functioning, they state that she likes to be around others and will take toys
from others and give toys to others. She enjoys affection.
With regards to communication, they say that she understands simple commands and can say
simple two-word sentences to make requests, such as, “want rice,” or “go to toilet.” However,
she is unable to carry on more complex conversation. She is able to communicate non-verbally
using nodding, head-shaking, waving, or taking others by the hand towards objects like the
television.
The parents deny significant repetitive behaviors. They say that she will occasionally repeat
other’s words but not do so over and over again. She does not repeat her own words. They deny
abnormal repetitive movements. They say she likes to play with a broad range of toys, especially
those that make a lot of noise. There were not reported sensitivities.
Past/Current Treatment: She is not in school and has not received services.
Past Medical History:
-Microcephaly -- noted since infancy.
Seizures -- Beginning at age 3 months, she began to have occasional seizures. Some were
reported as cyanosis around the mouth, others were reported as full body shaking. With
medications, she does not have seizures at present.
-Severe bilateral visual impairment – assessed to be central blindness
-History of left lower lobectomy -- status post bronchiectasis.
Medications: Phenobarbital
3
Social History: She lives at home with her parents and siblings in Saudi Arabia. She is cared for
by her parents and a nanny. She does not attend school.
Pregnancy/Birth Issues: The mother had no medical problems during pregnancy. There were no
exposures to substances or medications during the pregnancy.
Delivery: Normal vaginal delivery (NVD).
Full Term: Yes
Weight: 2.4kg
Immediate medical issues at birth: she was mildly cyanotic and per parents, she received nasal
oxygen but was not intubated. She had jaundice that was treated with light therapy. She was in
the hospital for 2 weeks after delivery.
Milestones: The parents had difficulty recalling precise ages but thought she began walking for
example, at approximately age 16 months. The parents recall delayed speech. For instance, she
started saying two-word sentences at age 3. There was no regression of any skills.
Clinical Observations:
In terms of adaptive behavior she could walk, sit, and explore objects with her hands.
Socially, she seemed to enjoy cuddling with mother. She was upset when parents left the room.
She smiled when interacted with. She requested her mother’s help with a pop-up toy. She took
objects from others and handed objects to others. She verbally requested information about the
activity of her sister who was present.
With regards to receptive language, she was able to follow-simple commands – like “give the toy
to mother.” However, she did not respond to bids at more complex conversation, like “how are
you doing?” Her expressive language was limited to simple two-word sentences, like “let’s
4
stop.” She could repeat simple words like, “doll.” Her prosody was odd. Non-verbally, she used
some appropriate gestures and smiled appropriately.
There were no definite stereotypies. She was observed humming to herself and rocking her head
back and forth while doing so, but this did not appear to be a stereotypical behavior.
In terms of her intellectual capacity, she could manipulate the levers of a simple pop-up toy, even
though she could not see the levers and demonstrated a slight understanding of the cause and
effect of the toy. She followed a simple command to give a doll to her mother.
Her appearance was notable for small head size. In addition, she looked pre-pubertal despite her
age.
Testing
Hearing Test: Reportedly normal per parents, results not available for review
IQ: Abnormally low per report, no record for review
Neuro-imaging:
CT scan of the Brain: 12/12/95 -- Normal non-contrast CT
Fragile X: Negative
Karyotyping: Normal
Metabolic Screening: Reportedly normal
Other Labs: Electrolytes within normal limits 8/2005
Ophthalmological Examination: Eye anatomy was reported to be within normal limits and a
diagnosis of central blindness, “brain blindness,” is given.
Anthropomorphic Data: at age of 15 years and 11 months
Height: 127 cm (<2.5 percentile) Saudi Growth Chart (El Mouzan, 2008). This corresponds to
CDC Z-Score of -5.5 or -5.5 standard deviations below the mean.
5
Weight: 20 kg (<2.5 percentile) Saudi Growth Chart (El Mouzan, 2008). This corresponds to
CDC z score of -14.94.
Head Circumference: 43.5cm (Greater than 3 standard deviations below the mean for Egyptian
girls) (Mean for Egyptian girls age 15 was 53.71cm. SD was 1.37) (Zaki et al., 2008).
National health and nutrition survey (NHANES), CDC/National Center for Health Statistics.
Multi-Axial Diagnosis:
Axis I: Intellectual Disability
Axis III: Microcephaly, Seizures, Bilateral Blindness, Short Stature, Delayed Puberty (Tanner
Stage-I according to her breast development), History of left lower lobectomy status post
bronchiectasis.
Axis IV: Multiple affected children
Patient IV:5
Age at Interview: 14
Patient IV:5 died when she was 18 years old because of chest infection (according to hospital’s
report).
HPI: The parents were first concerned medically when she had seizures starting at around age 3
months. The seizures involved jerking of the body, arms, and legs. They also noticed that she did
not reach her motor or language milestones on time. They feel that she was about 3 months
delayed in all of these.
In terms of social behavior, the parents say that she likes to play with other children. In addition,
she seeks and enjoys affection from her mother. She shows her toys to others. She does not play
imaginary games.
6
With regards to receptive language, the parents note that she can understand simple commands.
They add that they believe that her receptive language is stronger than her expressive language.
She is limited to two-word sentences like, “Want water,” and “Want car.” Her speech is
reportedly difficult for others to understand. Her parents say that she can wave goodbye, nod and
shake her head, and pull her mother towards the TV when she wants it on.
In terms of adaptive behavior, her parents say that she can feed herself and that she can dress
herself in a “messy” way. She cannot fully toilet herself although she does have bowel and
bladder control and can request to go to the bathroom. The parents say that she does not have the
intellectual capacity to attend school.
The parents do not note any regression of skills or language. There are no reported sensitivities
and no reported repetitive behaviors or narrowed interests.
Past/Current Treatment: She is currently receiving rehabilitative services where the focus is on
teaching her skills like how to feed herself and how to clothe herself.
Past Medical History:
-Microcephaly
-Seizures. They sound to be tonic-clonic. They started at age 3 months and have been well
controlled by medication but do recur occasionally.
-By report, she suffers from blindness bilaterally.
Medications: Phenobarbital.
Social History: She lives at home with her parents and siblings in Saudi Arabia. She is cared for
by her parents and a nanny. She does not attend school.
Pregnancy/Birth Issues: The mother had no medical problems during pregnancy. There were no
exposures to substances or medications during the pregnancy.
7
Delivery: NVD
Full Term: Yes
Weight: 2.4kg
Immediate medical issues after birth: She had physiologic jaundice that remitted without
treatment. She went home without hospitalization after birth. She fed normally on milk and
solids.
Milestones: The mother noted a three month delay in motor development across the board. She
says that there was a delay in language in that the patient only spoke a few single words at age
two.
Clinical Observations:
In terms of adaptive behavior and intellectual capacity, she could walk, sit, and explore objects
with hands. She was able to understand the levers of and manipulate a simple pop-up toy and
understood the cause and effect of all the different knobs and levers.
Socially, she was affectionate towards her mother. She smiled when interacted with. She took
objects from others and handed objects to others. She asked whether she should close certain
parts of the pop-up toy. She engaged in some imaginative play, rolling a car on the ground and
making a car sound in imitation of the examiner.
With regards to receptive language, she was able to follow-simple commands – like “give the
cars to mother.” She could reply to simple questions like, “What is this?” when a doll was put in
her hands. She would reply, “doll”. However, she did not respond to bids at more complex
conversation, like “how are you doing?” Her expressive language was limited to simple twoword sentences, like “Close it?” Non-verbally, she used some appropriate gestures and smiled
appropriately.
8
She exhibited some stereotypical movements such as flapping with both arms and some body
rocking.
Her appearance was notable for small head size. In addition, she looked pre-pubertal despite her
age.
Testing:
Hearing Test: Normal by parental report
Immunoglobulin levels (IgM, IgA, and IgG) were normal
Metabolic test for amino acid and fatty acid levels in both urine and blood were normal
Neuro-imaging:
MRI: 5/3/04 Per hospital report, “Bilateral occipital areas show increased signal intensity
in T2 with low intensity in T1 mainly in sub-cortical white matter. Prominent cortical
sulci and gyri of occipital lobes. Prominent occipital horn of lateral ventricle. Question of
vascular like ischemia or congenital metabolic leukodystrophy. No change since June 9,
2002.”
Fragile X: Negative
Karyotyping: Normal
Anthropomorphic Data: at age of 14
Height: 116 cm (<2.5 percentile) Saudi Growth Chart. (El Mouzan, 2008). This corresponds to
CDC Z score of -6.8 or -6.8 standard deviations from the mean.
Weight: 16.2 kg (<2.5 percentile) Saudi Growth Chart (El Mouzan, 2008). This corresponds to a
CDC z score of -11.74.
Head Circumference: 42.5cm (Greater than 3 standard deviations below the mean for Egyptian
girls) (Mean for Egyptian girls age 14 was 53.44cm. SD was 1.36) (Zaki et al., 2008).
9
Multi-Axial Diagnosis:
Axis I: Intellectual Disability
Axis III: Microcephaly, Seizures, Bilateral Blindness, Short Stature, Delayed Puberty (Tanner
Stage-I according to her breast development)
Axis IV: Multiple-affected children
Patient IV:6
Age at Interview: 6 years 11 months
HPI: The parents first brought the patient to medical attention when she started having seizures
at around age 3 months. They also noticed that she did not reach her motor or language
milestones on time.
In terms of social behavior, the parents say that she enjoys interacting with other people. She
smiles at them and likes to play with them. In addition, she seeks and enjoys affection from her
mother.
With regards to language, the parents note that she can say simple sentences. However, she
cannot engage in normal conversation. There was no report of echolalia.
In terms of adaptive behavior, her parents say that she can feed herself, dress herself, toilet
herself, open a book, and imitate cooking. They say that she does not have the intellectual
capacity to attend school.
The parents do not note any regression of skills or language. There are no reported sensitivities
and no reported repetitive behaviors or narrowed interests.
Past/Current Treatment: She has not received services.
Past Medical History:
10
-Microcephaly
-Seizures. They sound to be tonic-clonic. They started at age 3 months.
-By report, she suffers from severe, bilateral visual impairment
Medications: Phenobarbital.
Social History: She lives at home with her parents and siblings in Saudi Arabia. She is cared for
by her parents and a nanny. She does not attend school.
Pregnancy/Birth Issues: The mother had no medical problems during pregnancy. There were no
exposures to substances or medications during the pregnancy.
Delivery: Planned Cesarean Section
Full Term: Yes
Weight: Reportedly slightly lower than normal but not on file
Immediate medical issues after birth: the patient required supplemental oxygen at birth but was
no intubated. She was in the hospital for 3 weeks after delivery and required blood transfusions.
The cause for these is not reported. She went home without hospitalization after birth. She fed
normally on milk and solids.
Milestones: The mother noted a three month delay in motor development across the board. She
says that there was a delay in language in that the patient did not have words until age 2 years.
Clinical Observations:
In terms of adaptive behavior and intellectual capacity, she could walk, sit, and explore objects
with hands. She was able to understand and manipulate a simple pop-up toy but understood the
cause and effect of only one of several knobs.
11
Socially, she was affectionate towards her mother. She smiled when interacted with. She took
objects from others and handed objects to others. She asked whether it was ok to put a plastic
bottle of medicine into a cardboard box.
With regards to receptive language, she was able to follow-simple commands – like “give me the
car.” However, she did not respond to bids at more complex conversation, like “how are you
doing?” Her expressive language was limited to single words like “open” and “close.” Nonverbally, she used some appropriate gestures and expressions, smiling appropriately, for
example. At one point she brought a bag over to the examiner and presented it to express that she
would like it to be opened.
There were no repetitive behaviors or narrowed interests. Her appearance was notable for small
head size.
Testing
Hearing Test: Reportedly normal per parents
IQ: Not tested.
Neuro-imaging:
MRI Brain: April, 2004 Per hospital report, “Normal Axial T1,2”
EEG: Per hospital report, “Bad cortical organization,” “encephalopathy” but with epileptiform
activity.
Fragile X: Negative
Abnormal laboratory results: 6/16/2007 Liver Function Tests (LFT’s): Alk Phos 1296 (0-500),
ALT 106 (0-55), AST 121 (5-34).
Ophthalmologist Report: 1/13/08 “Slit lamp exam within normal limits.”
Anthropomorphic Data: at age of 6 years 11 months
12
Height: 100 cm (<2.5 percentile) Saudi Growth Chart. (El Mouzan, 2008). This corresponds to
CDC Z score of -4.4 or -4.4 standard deviations.
Weight: 12 kg (<2.5 percentile) Saudi Growth Chart (El Mouzan, 2008). This corresponds to a
CDC z-score of -6.24.
Head Circumference: 43cm (Greater than 3 standard deviations below the mean for Egyptian
girls) (Mean for Egyptian girls age 7 was 50.57cm. SD was 1.32) (Zaki et al., 2008).
Multi-Axial Diagnosis:
Axis I: Intellectual Disability
Axis III: Microcephaly, Seizures, Severe bilateral visual impairment, Short Stature
Axis IV: Multiple affected children
Patient IV:7
Age at Interview: 2 years and 1 month
HPI: The parents first brought him to medical attention at 3 months of age when he had seizures
that were described as his whole body shaking. Upon interview, they were concerned that the
patient was not reaching his motor or language milestones on time.
With regards to social behavior, they stated that he generally played by himself and liked toys
that made loud noises. In terms of language, the parents reported that he could say “mama,” and
“baba.” They said that he did not use non-verbal communication. He did not nod, shake his
head, or point, for example. With regards to adaptive skills at the time of the interview at age 2,
they said that he could walk but was not able to feed himself. There were no reported repetitive
behaviors, regression of skills, or sensitivities.
Past/Current Treatment: He has not received services.
13
Past Medical History:
-Seizures.
-Microcephaly
-He may suffer from bilateral visual impairment, by report
Medications: Phenobarbital
Social History: He lives at home with her parents and siblings in Saudi Arabia. She is cared for
by her parents and a nanny. He does not attend school.
Pregnancy/Birth Issues: The mother had no medical problems during pregnancy. There were no
exposures to substances or medications during the pregnancy.
Delivery: Cesarean Section, secondary to prolonged labor.
Full Term: Yes
Weight: 2.56kg
Immediate medical issues after birth: He had physiologic jaundice that lasted only 2-3 days and
that was treated with lights.
Milestones: The mother reports that he walked at 1 year and 8 months and that he only has two
single words at 2 years of age.
Clinical Observations:
In terms of adaptive behavior and intellectual capacity, he could walk, sit, and hold and explore
objects with his hands. He was able hold an object in each hand and to knock them together.
Socially, he smiled when interacted with and appeared to enjoy interacting with other people. He
attempted to hand a toy car to his mother and father.
With regards to receptive language, he was able to follow-simple commands – like “give the car
to your father.” He responded to his name, but not on the first time it was said. He did not
14
respond to bids at more complex conversation. His expressive language was limited to several
single words like “Mama,” “Baba,” and “No.” He could repeat words, like “car.” His main nonverbal communication was to smile at others and to hand them things.
There were no repetitive behaviors or narrowed interests. His appearance was notable for small
head size.
Testing
Hearing Test: None
IQ: None
Neuro-imaging:
MRI: 3/29/2009 Brain. T2 weighted Axial images showed; temporal pole atrophy with normal
cortical thickness and increased CSF space in the middle cranial fossa anterior to the temporal
pole, dilatation of the lateral ventricles, normal third ventricular size and the atria of the lateral
ventricles, small splenium of the corpus callosum, normal appearance of the optic nerves and
chiasm. The sylvian fissures appear normal in size and symmetry with normal configuration.
T1 sagittal images confirmed atrophy of the temporal lobe most prominent at the pole and the
dilatation of the lateral ventricle. Midline sagittal T1 shows hypoplasia of the rostrum and
splenium of the corpus callosum. Aqueduct and fourth ventricle are normal in size.
Coronal FLAIR images confirmed the normal appearance of the chiasm (anterior to the temporal
horns of the lateral ventricles).
Ophthalmology: 2-17-09 and 5/1/2009 normal slit lamp and funduscopic examinations.
Fragile X: Negative
Metabolic Screening: National newborn screening HPLC 5/24/09 – Arginine H 406 (16-180),
Alanine L 87 (120-720), otherwise normal.
15
Anthropomorphic Data: at age of 2 years and 1 month
Height: 85 cm (25th percentile) Saudi Growth Chart (El Mouzan, 2008). This corresponds to
CDC z score of -0.5 or -0.5 standard deviations.
Weight: 10.2 kg (10th percentile) Saudi Growth Chart (El Mouzan, 2008). This corresponds to a
CDC z-score of -2.11.
Head Circumference: 41.5cm (Substantially below the 3rd percentile) Source:
Head
circumference-for-age percentiles, boys birth to 36 months, CDC growth charts
Multi-Axial Diagnosis:
Axis I: Intellectual Disability
Axis III: Microcephaly, Seizures, Possible visual impairment
Axis IV: Multiple affected children
Patient IV:8
Age at Interview: 3 months and 3 weeks old
HPI: The patient was brought to medical attention at 3 months of age when he had abnormal eye
movements his parents thought were seizures.
Past Medical History:
-Microcephaly
-Possible visual impairment, by observation
Medications: Phenobarbital 30 mg a day
Social History: He lives at home with his parents and siblings in Saudi Arabia. He is cared for
by his parents.
16
Pregnancy/Birth Issues: The mother had no medical problems during pregnancy. There were no
exposures to substances or medications during the pregnancy.
Delivery: Cesarean Section
Full Term: Yes
Weight: 2,700 grams
Immediate medical issues after birth: He had physiologic jaundice that lasted only one week and
that was treated with lights.
Milestones: Delayed milestones
Clinical Observations: He does not appear to see objects in his environment or light.
Ophthalmology: An appointment is pending.
Metabolic Screening: National newborn screening results are pending
Anthropomorphic Data: 3 months and 3 weeks old.
Height: Not available.
Weight: 5 kg (3th percentile) Saudi Growth Chart (El Mouzan, 2008). This corresponds to a CDC
z-score of between -2 and -1.5.
Head Circumference: 32 cm. Greater than 3 standard deviations below the mean for Egyptian
boys) (Mean for Egyptian boys age 4 months was 41.58cm. SD was 1.14) (Zaki et al., 2008).
Multi-Axial Diagnosis:
Axis I: Intellectual Disability
Axis III: Microcephaly, Seizures, Severe bilateral visual impairment, Short Stature
Axis IV: Multiple affected children
17
Supplemental Figures
Supplementary Figure 1. Genome-wide parametric linkage analysis for SAR1008 pedigree.
Plot shows allele sharing LOD (Logarithm of Odds) on Y axis and chromosome location on X
axis. The red horizontal line indicates the maximum theoretical LOD score for this family (3.7).
The vertical dashed lines show the boundaries of each chromosome.
18
Supplementary Figure 2. The amino acid sequence alignment of DIAPH1 protein across
species using ClustalW2 program and nonsense homozygous mutation in DIAPH1. (A) The
site of p.Gln778* mutation (Q778*) is shaded with yellow. An asterix (*) highlights identical
residues. (B) A nonsense mutation in the DIAPH1 is identified through custom designed target
sequencing. Sanger sequencing of cDNA from lymphoblastoid cells confirms this mutation. The
altered amino-acid sequence leading to a premature stop-codon (p.Gln778*) is shown in red.
19
Supplementary Figure 3. DIAPH1 Expression Pattern in Human and in embryonic and
adult mouse brains. (A) In situ hybridization for Diap1 mRNA in E12.5, E14.5, and E17.5
mouse brains. Antisense diap1 riboprobes labeled with digoxigenin-UTP were used to detect
Diap1 mRNA. Diap1 mRNA was detected across brain regions such as the developing cerebral
cortex, ventral forebrain, lateral midbrain, LGE (lateral ganglionic eminences) and MGE (medial
ganglionic eminences) in E12.5, E14.5. In situ hybridization for Diap1 mRNA in E17.5 brains
showed expression in the cortical plate, the developing hippocampus, and basal ganglia. Scale
bars, 500 µm at lower magnification and 100 µm at higher magnification. (B) In situ
hybridization for Diap1 mRNA during postnatal development on P3, P7, and P14 in mouse
brain. Diap1 mRNA was detected in differentiating neurons cortical plate, hippocampus,
thalamus, basal ganglia, and external layer of cerebellum. Scale bars, 500 µm.
20
Supplementary Figure 4. Gene co-expression analysis revealed co-expressed genes with
DIAPH1 and previously identified microcephaly genes (MCPH1, WDR62, CDK5RAP2, ASPM),
CENPJ, STIL, CEP135). The software Cytoscape was used to visualize the co-expression
network, in which seed microcephaly genes are shown by circles and the correlated genes are
connected by lines.
21
Supplementary Figure 5. mDia1 expression in the whole brain lysates from wild-type (+/+),
and homozygous (–/–) mice of Diap1 genotype. GAPDH was used as an internal control. Global
appearance of Diap1-/- was similar to Diap1+/+ mouse.
22
Supplementary Figure 6. mDia1-KO mice show preserved layer organization. Coronal
sections of Wt and mDia1-KO mice at P0 were stained with cortical layer specific markers;
CTIP2, Layer V (green) and CUX1, Layers II-IV (red). Scale bars 1mm at lower magnification
and 50µm at higher magnification.
23
Supplementary Figure 7. Coronal sections of brain from Wt and mDia1-KO mice were
stained with NisslNote that no blockage of cerebral aqueduct of sylvius is detected. Scale bar
1mm.
24
Supplementary Figure 8. Immunostaining for adherent junction components. β-catenin,
phalloidin (Upper panel); N-Cadherin,
E-cadherin (Middle panel); and α-tubulin, γ-tubulin
(Lower panel) in coronal sections of lateral vertical wall from Wt and mDia1-KO-Mouse at
E14.5 day. Nuclei were counterstained with DAPI (blue). All images shown at 50µm
magnification.
25
Supplementary Figure 9. Immunostaining for GABA. Coronal sections of the cerebral cortex
from mDia1-KO and Wt at P0 were used. Images shown at 200µm magnification.
26
Supplementary Figure 10. Corpus callosum measurement. The thickness of the corpus
callosum was measured in Diap1+/+ and Diap1 -/- at the rostral septal area. Images shown at 100
µm magnification.
27
Supplementary Table 1. IBD estimates for parents and siblings. To detect familial relationship within the family, pairwised IBD
estimate was calculated using the parameters by comparing 40 Arabic ethnicity samples [Independent minor allele frequency is >5%,
Hardy Weinberg eq. p=0.0001, 100% call rate, R2 (SNP correlation coefficient) is 0.23]
IID1a
IID2b
EZc
Z0d
Z1e
Z2f
PI_HATg
DSTh
PPCi
RATIOj
1008-01 (Father)
1008-02 (Mother)
0
0.7379 0.2292 0.0329
0.1475 0.706989
1
2.6079
1008-05 (Affected)
1008-07 (Affected)
0.5
0.1448 0.4704 0.3848
0.62
0.852272
1
16.7247
1008-02 (Mother)
1008-04 (Affected)
0.5
0.0006 0.8667 0.1328
0.5661
0.81598
1
NA
1008-03 (Affected)
1008-05 (Affected)
0.5
0.2143 0.4485 0.3371
0.5614
0.83387
1
10.1389
1008-02 (Mother)
1008-07 (Affected)
0.5
0.001
0.8835 0.1155
0.5573 0.812276
1
1480
1008-01 (Father)
1008-03 (Affected)
0.5
0.0008 0.8967 0.1026
0.5509 0.809552
1
1533.5
1008-01 (Father)
1008-05 (Affected)
0.5
0.0006 0.8995 0.0999
0.5497 0.809013
1
3018
1008-01 (Father)
1008-07 (Affected)
0.5
0.0008
0.903
0.0962
0.5477 0.808213
1
NA
1008-01 (Father)
1008-06 (Normal)
0.5
0.0004 0.9119 0.0877
0.5436 0.806442
1
NA
1008-02 (Mother)
1008-07 (Affected)
0.5
0.0006 0.9127 0.0868
0.5431 0.806226
1
NA
1008-02 (Mother)
1008-05 (Affected)
0.5
0.0014 0.9114 0.0873
0.543
0.806244
1
978.3333
1008-02 (Mother)
1008-03 (Affected)
0.5
0.001
0.9168 0.0822
0.5406 0.805219
1
996.3333
1008-03 (Affected)
1008-07 (Affected)
0.5
0.2373 0.4526 0.3101
0.5364 0.825392
1
9.5311
1008-04 (Affected)
1008-05 (Affected)
0.5
0.2435 0.4436 0.3129
0.5347 0.825249
1
9.7993
1008-03 (Affected)
1008-04 (Affected)
0.5
0.2433 0.4665 0.2901
0.5234 0.820448
1
8.9937
1008-01 (Father)
1008-04 (Affected)
0.5
0.0006 0.9529 0.0465
0.523
0.797695
1
2935
1008-04 (Affected)
1008-06 (Normal)
0.5
0.2457 0.5134 0.2409
0.4976 0.809732
1
7.727
1008-05 (Affected)
1008-06 (Normal)
0.5
0.2629 0.4893 0.2479
0.4925 0.809166
1
7.5627
1008-04 (Affected)
1008-07 (Affected)
0.5
0.258
0.506
0.236
0.489
0.807224
1
9.0489
1008-03 (Affected)
1008-06 (Normal)
0.5
0.2621 0.5189 0.2191
0.4785 0.803143
1
7.2581
1008-06 (Normal)
1008-07 (Affected)
0.5
0.3493 0.4376 0.2131
0.4319 0.791501
1
5.4013
a
b
c
IID1; Individual ID for first individual, IID2; Individual ID for second individual, Expected IBD sharing given PED file, dZ0;
P(IBD=0,eZ1; P(IBD=1),fZ2; P(IBD=2), gPI_HAT; P(IBD=2)+0.5*P(IBD=1) (proportion IBD), hDST; IBS (pairwise identity-by-state
(IBS)) distance (IBS2 + 0.5*IBS1)/(N SNP pairs ), iPPC; IBS binomial test Ratio of het; j RATIO; IBS 0 SNPs (expected value is 2)
28
Supplementary Table 2. Genotyping data from Illumina IMv1 Duo Bead array chip shows the
homozygous block across the affected and unaffected members of the pedigree on chromosome
5. Linkage interval is highlighted with yellow.
rs Number
rs10463951
rs2042243
rs6872555
rs4270686
rs4423993
rs2188464
rs4976526
rs12188192
rs2348188
rs1434660
rs2961624
rs1229742
rs739699
rs1528961
rs10491336
rs2189082
rs2057680
rs11242395
rs2967806
rs217259
rs12516561
rs10515498
rs10040792
rs4835696
rs7379760
rs4835646
rs1651031
rs904612
rs1800954
rs197197
rs6580353
rs155939
rs1623177
rs269783
rs2436396
rs7268
rs6872579
rs2286394
rs3756325
rs702390
rs2907308
rs10491311
rs3749765
rs2237079
rs251177
rs2306339
rs3763120
rs349132
cM
138.18
138.1851
138.2819
138.3226
138.3292
138.4628
138.5062
138.8296
138.844
138.8562
138.8791
138.9029
138.9083
138.9186
138.9405
138.9431
138.9516
138.9598
138.96
138.96
138.96
138.96
138.96
139.38
139.38
139.38
139.38
139.38
139.4812
139.5516
139.5935
139.6114
139.6271
139.6645
139.6768
139.7982
139.8412
140.1294
140.5398
140.6337
140.64
140.64
140.64
140.64
140.64
140.64
140.64
140.64
III:1
BB
AB
AB
AB
AB
BB
AB
AB
AB
BB
BB
AA
AA
BB
BB
BB
AB
BB
AA
AA
BB
AB
AB
AB
AA
AA
AB
BB
AA
AB
BB
AB
BB
AB
BB
AA
AB
BB
BB
BB
BB
AB
AA
AB
AB
BB
AB
AB
III:2
AA
AA
AA
BB
BB
BB
AA
BB
AB
BB
BB
AA
AA
BB
BB
BB
AA
BB
AB
AA
BB
AB
AB
BB
AA
AA
AA
AB
AA
AB
BB
BB
BB
AB
BB
AB
AA
BB
BB
BB
AB
BB
AB
AA
BB
BB
AA
AB
IV:2
AB
AB
AB
AB
AB
BB
AB
AB
BB
BB
BB
AA
AA
BB
BB
BB
AB
BB
AB
AA
BB
BB
BB
AB
AA
AA
AB
AB
AA
BB
BB
AB
BB
AA
BB
AB
AB
BB
BB
BB
AB
AB
AB
AB
AB
BB
AB
AA
IV:4
AB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
AA
BB
AA
AA
BB
AA
AA
AA
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
29
IV:5
AB
AA
AA
BB
BB
BB
AA
BB
AB
BB
BB
AA
AA
BB
BB
BB
AA
BB
AB
AA
BB
AB
AB
BB
AA
AA
AA
AB
AA
AB
BB
BB
BB
AB
BB
AB
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
IV:6
AB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
AA
BB
AA
AA
BB
AA
AA
AA
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
IV:7
AB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
AA
BB
AA
AA
BB
AA
AA
AA
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
IV:8
AB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
AA
BB
AA
AA
BB
AA
AA
AA
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
rs32929
rs10515511
rs32927
rs32946
rs152355
rs248504
rs3815355
rs2974704
rs10063472
rs6888135
rs351260
rs164078
rs164495
rs6580215
rs252144
rs13185299
rs2288819
rs10072521
rs2908528
rs11167773
rs959662
rs254441
rs153423
rs4912639
rs17208817
rs10491399
rs249740
rs4244029
rs152422
rs13179022
rs34016
rs152528
rs2070715
rs388940
rs2436379
rs10044036
rs3776331
rs246599
rs258799
rs258770
rs2074637
rs3776221
rs1438734
rs853158
rs10482672
rs2963156
rs7701443
rs4912913
rs2121152
rs4912922
rs7712869
rs888993
rs153516
rs12657648
rs2910256
rs1427864
140.64
140.64
140.64
140.64
140.64
140.64
140.64
140.653
140.7045
140.724
140.8706
140.9472
140.9868
141.097
141.2112
141.4377
141.497
141.7078
141.7211
141.8135
141.9159
142.0658
142.1954
142.2689
142.301
142.3418
142.4064
142.4553
142.5406
142.6088
142.6816
142.7312
142.748
143.1474
143.1953
143.3101
143.8453
144.0535
144.077
144.1599
144.2253
144.2354
144.2522
144.2747
144.5042
144.6776
144.7673
144.8348
144.9234
145.0013
145.1302
145.1609
145.2575
145.377
145.4029
145.4091
AB
AA
BB
AB
AB
AA
AA
BB
BB
AB
AB
AA
AB
AA
AB
AA
AB
BB
BB
BB
BB
AA
AA
AB
AB
BB
AA
BB
BB
AB
AB
AA
AB
AA
AA
BB
BB
AB
BB
AB
AA
AA
AB
AA
BB
BB
AB
AA
AB
AB
BB
AA
AB
AB
AB
AA
AB
AA
BB
AB
BB
AA
AB
BB
BB
AB
BB
AB
AA
AA
AA
AA
BB
BB
BB
BB
BB
AA
AB
AB
AB
BB
AA
BB
BB
BB
BB
AB
AB
AA
AA
BB
AB
AA
BB
BB
AA
AA
BB
AA
BB
BB
AB
AB
AA
AB
BB
AB
AB
AB
BB
AB
AA
AA
BB
BB
AB
AA
AB
BB
BB
AA
AB
AB
AB
AA
AB
AA
AB
BB
BB
BB
BB
AA
AB
BB
AA
BB
AA
BB
BB
AB
AB
AB
BB
AA
AA
BB
AB
AB
BB
AB
AA
AA
AB
AA
BB
BB
BB
AB
AB
AA
BB
AB
AA
BB
AB
AB
BB
AA
BB
AA
BB
AA
AA
BB
BB
BB
BB
AA
AA
AA
AA
AA
BB
BB
BB
BB
BB
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
AA
BB
BB
AA
AA
AA
BB
BB
AA
BB
AA
BB
AA
30
BB
AA
BB
AA
BB
AA
AA
BB
BB
BB
BB
AA
AA
AA
AA
AA
BB
BB
BB
BB
BB
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
AA
BB
BB
AA
AA
AA
BB
BB
AA
BB
AA
BB
AA
BB
AA
BB
AA
BB
AA
AA
BB
BB
BB
BB
AA
AA
AA
AA
AA
BB
BB
BB
BB
BB
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
AA
BB
BB
AA
AA
AA
BB
BB
AA
BB
AA
BB
AA
BB
AA
BB
AA
BB
AA
AA
BB
BB
BB
BB
AA
AA
AA
AA
AA
BB
BB
BB
BB
BB
AA
AB
AB
AB
BB
AA
BB
BB
BB
BB
AB
AB
AA
AA
BB
AB
AA
BB
BB
AA
AA
BB
AA
BB
BB
AB
AB
AA
AB
BB
AB
AB
AB
BB
AB
BB
AA
BB
AA
BB
AA
AA
BB
BB
BB
BB
AA
AA
AA
AA
AA
BB
BB
BB
BB
BB
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
AA
BB
BB
AA
AA
AA
BB
BB
AA
BB
AA
BB
AA
rs1366084
rs867924
rs1024961
rs7718152
rs1421776
rs318373
rs4513726
rs6580315
rs723223
rs3910188
rs724382
rs17102282
rs2163748
rs9324976
rs13359856
rs10515549
rs1363556
rs1368434
rs340051
rs970456
rs886949
rs758037
rs11435
rs6881180
rs1201598
rs9325005
rs7718446
rs11747475
rs4705334
rs9986288
rs986061
rs322487
rs2400228
rs10068414
rs1480158
rs720305
rs2053030
rs31036
rs31039
rs11956052
rs2400293
rs17106769
rs2194156
rs1432830
rs6895278
rs994025
rs1025489
rs11958481
rs721570
rs6580511
rs2080085
rs2303064
rs7723153
rs888956
rs11742884
rs12654778
145.4305
145.5426
145.7954
145.8694
145.9689
146.0901
146.2076
146.2661
146.363
146.55
146.55
146.55
146.56
146.56
146.5861
146.67
146.67
146.862
147.1864
147.2798
147.3661
147.4216
147.4941
147.7117
147.8936
147.9406
147.9511
148.0037
148.0596
148.1111
148.2134
148.3359
148.4269
148.7726
148.9792
149.0082
149.2827
149.3485
149.3526
149.4635
149.4997
149.7836
149.949
150.03
150.03
150.03
150.03
150.03
150.03
150.0575
150.0969
150.2711
150.8526
151.2071
151.3001
151.3322
AA
BB
AB
BB
AB
AB
AB
BB
AB
BB
AB
BB
AB
BB
AA
AA
BB
AA
AA
AA
AB
AA
BB
BB
AA
AA
AA
AA
AA
AA
AB
AB
BB
BB
BB
AB
BB
AA
AA
AA
AA
AB
BB
AA
AB
BB
BB
AB
AA
AA
AB
BB
AA
AB
AA
AA
AA
BB
BB
BB
AA
AB
BB
AB
BB
BB
BB
AB
BB
AB
AA
AA
BB
AA
AA
AA
BB
AB
BB
BB
AA
AA
AA
AB
AB
AA
AA
AB
AB
BB
BB
AB
BB
AB
AA
AA
AA
AB
BB
AA
AB
BB
BB
AB
AB
AA
BB
BB
AA
BB
AA
AB
AA
BB
AB
BB
AB
AA
AB
AB
AB
BB
AB
AB
AB
AB
AA
AA
BB
AA
AA
AA
AB
AB
BB
BB
AA
AA
AA
AB
AB
AA
AB
AA
AB
BB
BB
BB
BB
AB
AA
AA
AA
AA
BB
AA
AA
BB
BB
AA
AB
AA
AB
BB
AA
AB
AA
AB
AA
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
BB
AA
BB
BB
AA
AA
AA
AA
AA
AA
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
AA
AA
31
AA
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
BB
AA
BB
BB
AA
AA
AA
AA
AA
AA
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
AA
AA
AA
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
BB
AA
BB
BB
AA
AA
AA
AA
AA
AA
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
AA
AA
AA
BB
BB
BB
AA
AB
BB
AB
BB
BB
BB
AB
BB
AB
AA
AA
BB
AA
AA
AA
BB
AB
BB
BB
AA
AA
AA
AB
AB
AA
AA
AB
AB
BB
BB
AB
BB
AB
AA
AA
AA
AB
BB
AA
AB
BB
BB
AB
AB
AA
BB
BB
AA
BB
AA
AB
AA
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
BB
AA
BB
BB
AA
AA
AA
AA
AA
AA
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
BB
BB
AA
BB
BB
BB
BB
AA
AA
BB
BB
AA
BB
AA
AA
rs3857420
rs11957757
rs4705286
rs4705064
rs994446
rs36077
rs4147470
rs9325132
rs1438692
rs1623904
rs1659091
rs10515626
rs352336
rs813035
rs6887452
rs2042249
rs476741
rs2431718
rs241280
rs7719315
rs1056993
rs2279132
rs6866304
rs6874724
rs728837
rs2061765
rs1422429
rs10875551
rs30825
rs173438
rs245076
rs216128
rs216148
rs216124
rs13362413
rs4358508
rs980272
rs919741
rs4958445
rs888714
rs253296
rs1560657
rs752922
rs253345
rs33412
rs2054440
rs2042235
rs3828599
rs3792783
rs3792780
rs999556
rs17659044
rs1012415
rs10515644
rs1862362
rs10515645
151.3429
151.3475
151.4739
151.4904
151.5408
151.663
151.8035
151.8326
151.9959
152.0083
152.0186
152.0559
152.0672
152.0842
152.1773
152.1992
152.2233
152.2788
152.2907
152.3736
152.5058
152.5134
152.5323
152.5568
152.5634
152.5841
152.7369
152.7467
152.922
152.9869
153.0062
153.1211
153.1464
153.1833
153.2544
153.4464
153.6918
153.7473
153.8478
155.1285
155.1455
155.2285
155.2307
155.2817
155.3186
156.0642
156.0917
156.0999
156.2143
156.2365
156.2524
156.3176
156.324
156.3632
156.3733
156.4182
BB
BB
AB
BB
AB
AB
BB
AB
AB
AB
AB
AB
AB
AB
AA
BB
AB
AB
AB
AA
AB
BB
AB
BB
AA
BB
AB
AB
AA
BB
AB
AB
AB
BB
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
AB
AA
AB
AA
AB
BB
BB
AB
AB
AB
AB
AB
BB
AB
AB
AB
AA
AB
BB
AB
AA
AB
AB
AB
AB
AA
AB
BB
AB
BB
AB
BB
AA
AB
AB
BB
AA
AA
AA
BB
BB
AA
BB
BB
AB
AA
BB
AB
BB
AA
BB
AA
AB
BB
AA
AA
AB
AA
AB
AA
AB
BB
BB
AB
AA
AB
BB
BB
BB
BB
BB
BB
AB
BB
AB
AA
AA
AB
BB
BB
AA
AA
AA
BB
AA
BB
AB
BB
AB
BB
AB
BB
AB
AB
AB
BB
BB
AA
BB
BB
AB
AA
BB
AB
BB
AA
BB
AA
AB
BB
AA
AA
AA
AA
BB
AA
AA
BB
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
AA
AA
BB
BB
AA
BB
AA
AA
BB
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
BB
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
AA
AA
AA
BB
BB
32
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
AA
AA
BB
BB
AA
BB
AA
AA
BB
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
BB
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
AA
AA
AA
BB
BB
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
AA
AA
BB
BB
AA
BB
AA
AA
BB
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
BB
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
AA
AA
AA
BB
BB
BB
AB
AB
AB
AB
AB
BB
AB
AB
AB
AA
AB
BB
AB
AA
AB
AB
AB
AB
AA
AB
BB
AB
BB
AB
BB
AA
AB
AB
BB
AA
AA
AA
BB
BB
AA
BB
BB
AB
AA
BB
AB
BB
AA
BB
AA
AB
BB
AA
AA
AB
AA
AB
AA
AB
BB
BB
BB
BB
BB
AA
AA
BB
AA
AA
AA
AA
AA
BB
BB
AA
BB
AA
AA
BB
AA
BB
BB
BB
BB
AA
BB
AA
AA
AA
BB
AA
AA
AA
BB
BB
AA
BB
BB
AA
AA
BB
BB
BB
AA
BB
AA
BB
BB
AA
AA
BB
AA
AA
AA
BB
BB
rs448503
rs153470
rs1991798
rs153445
rs7717132
rs246498
rs2431079
rs165359
rs2053028
rs3097775
rs2304054
rs7736409
rs2278383
rs1368368
rs2033467
rs4958487
rs890832
rs302412
rs170020
rs10515655
rs294958
rs918462
rs6875501
rs2961739
rs7702336
rs1010100
rs1438940
rs10515679
rs1438946
rs2973139
rs1871164
rs4958655
rs578772
rs548294
rs12522802
rs778821
rs1493383
rs1422884
rs729329
rs11741511
rs1461234
rs286969
rs11744052
rs889032
rs3886611
rs153411
rs6870010
rs3172941
rs10037652
rs283438
rs9324786
rs12374480
rs4958756
rs13165424
rs1319694
156.4499
156.5169
156.5608
156.6336
156.6359
156.6907
156.7806
157.0741
157.1602
157.2276
157.2481
157.2942
157.3102
157.3148
157.4529
157.504
157.9423
158.0749
158.0873
158.1718
158.261
158.3966
158.4333
158.44
158.44
158.44
158.44
158.44
158.44
158.44
158.4698
158.5243
158.6071
158.6091
158.7019
158.74
158.7644
158.7874
158.81
158.8493
159.0875
159.1845
159.48
159.48
159.5015
159.5102
159.5186
159.5413
159.5756
159.5937
159.6138
159.6701
159.6969
159.881
159.9439
BB
AB
BB
AB
BB
AA
BB
BB
BB
AB
BB
AB
BB
BB
AA
AB
AA
AA
AA
AA
AA
AB
BB
BB
BB
AA
AB
AB
AB
AB
BB
AA
AB
AB
AA
BB
BB
AA
BB
BB
AB
AA
BB
AB
AA
BB
AB
AB
BB
BB
BB
AA
AA
AA
BB
BB
AB
BB
BB
BB
AB
BB
BB
BB
BB
BB
BB
BB
AB
AB
AB
AA
AB
AA
AB
AB
AB
BB
BB
AB
AB
AA
BB
AA
AA
AB
AB
BB
AB
AA
AB
AB
AB
BB
AB
AB
AB
BB
AB
AB
BB
AA
AA
BB
BB
BB
AA
AB
AA
AA
BB
AA
BB
AB
BB
AB
BB
BB
BB
AB
BB
AB
BB
AB
AB
AA
AA
AB
AA
AB
AB
BB
BB
BB
AB
AB
AB
AB
AB
AB
AB
AB
AB
AA
AA
AB
AB
AB
BB
AB
AA
AB
BB
BB
AB
BB
AB
AB
BB
BB
BB
AA
AB
AA
AB
BB
BB
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
AA
AA
BB
BB
BB
AA
AA
BB
AA
AA
BB
AA
BB
BB
AA
BB
BB
AA
BB
BB
BB
AA
BB
AA
AA
BB
AA
AA
BB
BB
BB
AA
AA
AA
AB
33
BB
BB
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
AA
AA
BB
BB
BB
AA
AA
BB
AA
AA
BB
AA
BB
BB
AA
BB
BB
AA
BB
BB
BB
AA
BB
AA
AA
BB
AA
AA
BB
BB
BB
AA
AA
AA
AB
BB
BB
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
AA
AA
BB
BB
BB
AA
AA
BB
AA
AA
BB
AA
BB
BB
AA
BB
BB
AA
BB
BB
BB
AA
BB
AA
AA
BB
AA
AA
BB
BB
BB
AA
AA
AA
AB
BB
AB
BB
BB
BB
AB
BB
BB
BB
BB
BB
BB
BB
AB
AB
AB
AA
AB
AA
AB
AB
AB
BB
BB
AB
AB
AA
BB
AA
AA
AB
AB
BB
AB
AA
AB
AB
AB
BB
AB
AB
AB
BB
AB
AB
BB
AA
AA
BB
BB
BB
AA
AB
AA
AB
BB
BB
BB
BB
BB
AA
BB
BB
BB
BB
BB
BB
BB
BB
AA
BB
AA
AA
AA
AA
AA
AA
BB
BB
BB
AA
AA
BB
AA
AA
AB
AB
BB
AB
AA
AB
AB
AB
BB
AB
AB
AB
BB
AB
AB
BB
AA
AA
BB
BB
BB
AA
AB
AA
AB
Supplementary Table 3. Coverage distributions and error rates across the targeted
Whole Exome
Sequencing
Targeted
homozygosity
Interval
homozygosity interval
Number of lanes
Read Type
Read length
Total number of reads (millions)
% mapped to the genome
Interval size (bp)
% mapped to the interval
Mean Coverage
% of bases covered at least 4X
Mean error rate (%)
2nd base error rate (%)
Last base error rate (%)
Total number of reads (millions)
% mapped to the genome
% mapped on target
Mean Coverage
% of bases covered at least 4X
Mean error rate (%)
2nd base error rate (%)
Last base error rate (%)
IV:4
(proband)
III:1 (father)
1
Single read
74
19.81
96.40
998,145
71.68
42.57
90.60
0.48
0.51
0.78
88.3
92.26
67.29
91.7
97.54
0.43
0.49
1.22
1
Single read
74
19.65
96.89
998,145
75.42
47.56
91.21
0.48
0.51
0.91
34
Supplementary Table 4. Variants identified within the homozygous linkage interval on
chromosome 5
Start (hg19)
Genomic
Location
Gene
AA_Change
bp_Change
dbSNP
140724060
g.460A>C
PCDHGA3
p.I154L
c.460A>C
rs11575948
140725828
g.2228C>T
PCDHGA3
p.A743V
c.2228C>T
rs7736541
140730384
g.557G>T
PCDHGB1
p.S186I
c.557G>T
rs62378414
140731022
g.1195A>G
PCDHGB1
p.K399E
c.1195A>G
rs77250251
140735215
g.448G>A
PCDHGA4
p.A150T
c.448G>A
rs11575949
140741738
g.2036G>C
PCDHGB2
p. R679P
c. 2036G>C
rs62378417
140741761
g.2059A>G
PCDHGB2
p.K687E
c.2059A>G
rs57735633
140750044
g.83T>C
PCDHGB3
p.V28A
c. 83T>C
rs6860609
140751128
g.1167C>G
PCDHGB3
p.N389K
c.1167C>G
rs2240697
140751696
g.1735C>A
PCDHGB3
p.P579T
c.1735C>A
rs62620756
140763029
g.563A>G
PCDHGA7
p.E188G
c.563A>G
rs2072315
140789933
g.2164G>A
PCDHGB6
p.A722T
c.2164G>A
rs3749767
140953085
g.45,397C>T
DIAPH1
p.Q778*
c.2332C>T
novel
141019110
g.1318C>A
RELL2
p.L133I
c.397C>A
rs14251
141021096
g.9840C>T
FCHSD1
p.P681L
c.2042C>T
rs32957
141336264
g.1153C>A
PCDH12
p.H385N
c.1153C>A
rs164075
35
Minor Allele
Frequencies
C: 12.335%
(392 / 3178)
C: 47.208%
(1040 / 2203)
T: 2.466% (54 /
2190)
G: 2.558% (59 /
2306)
A: 12.615%
(330 / 2616)
C: 11.789%
(325.155 /
2758)
G: 12.489%
(274 / 2194)
T: 0.086% (3 /
3476)
C: 46.002%
(1398 / 3039)
A: 3.105% (68 /
2190)
A: 46.040%
(2215 / 4811)
A: 30.564%
(1343 / 4394)
A: 43.414%
(3764 / 8670)
T: 3.655% (138
/ 3776)
C: 22.988%
(2279 / 9914)
Supplementary Table 5. Primary and secondary antibodies used for immunostaining
Primer Antibodies and dilutions
Company
Rabbit anti-Cux1 (1:250)
Santa Cruz-sc13024
Rat anti-Ctip2 (1:250)
Santa Cruz-sc56014
Rabbit anti-TBR1 (1:500)
Abcam-ab31940
Mouse anti beta-cathenin (1:250)
BD Laboratories-610153
Rabbit anti-N-cadherin (1:250)
Santa Cruz-sc7939
Mouse anti E-cadherin (1:250)
BD Laboratories-610405
Alpha-Tubulin FITC-conjugated (1:500)
Sigma-F2168
Rabbit anti-gamma-tubulin (1:250)
Sigma-T5192
Texas Red®-X Phalloidin (1:500)
Invitrogen-T7471
Rabbit anti-GABA (1:5000)
Sigma-A2052
Rabbit anti-Pax6 (1:300)
BD Pharmingen-61462
Secondary Antibodies
Alexa Fluor® 488 Donkey Anti-Mouse IgG (H+L) (1:1000)
Invitrogen-A21202
Alexa Fluor® 488 Donkey Anti-Rabbit IgG (H+L) (1:1000)
Invitrogen-A21206
Alexa Fluor® 488 Donkey Anti-Rat IgG (H+L) (1:1000)
Invitrogen-A21208
Alexa Fluor® 594 Donkey Anti-Rabbit IgG (H+L) (1:1000)
Invitrogen-A21207
Alexa Fluor® 594 Donkey Anti-Mouse IgG (H+L) (1:1000)
Invitrogen-A21203
36
Supplemental References
El Mouzan, M. I., Al Salloum, Abdullah A., Al Herbish, Abdullah S., Quarashi, Mansour M., Al
Omar., Ahmad A. (2008). Health Profile for Saudi Children and Adolescents (No. AR-20-63)
(Riyadh, King Abdulaziz City for Science & Technology), pp. 96.
Zaki, M. E., Hassan, N. E., and El-Masry, S. A. (2008). Head circumference reference data for
Egyptian children and adolescents. East Mediterr Health J 14, 69-81.
37